This invention relates generally to hematopoietic cells, and more specifically to methods and devices for long-term in vitro culturing of hematopoietic progenitor cells, as well as methods for the introduction of exogenous genetic material into cells of hematopoietic origin.
The circulating blood cells, such as erythrocytes, leukocytes, platelets and lymphocytes, are the products of the terminal differentiation of recognizable precursors. In fetal life, hematopoiesis occurs throughout the reticular endothelial system. In the normal adult, terminal differentiation of the recognizable precursors occurs exclusively in the marrow cavities of the axial skeleton, with some extension into the proximal femora and humeri. These precursor cells, in turn, derive from very immature cells, called progenitors, which are assayed by their development into contiguous colonies of mature blood cells in 1-3 week cultures in semi-solid media, such as methylcellulose.
There have been reports of the isolation and purification of hematopoietic progenitor cells (see, e.g., U.S. Pat. No. 5,061,620 as representative), but such methods have not allowed for the long-term culture of such cells that maintain their viability and pluripotency.
Studies of the murine hematopoietic system in the murine bone marrow have resulted in a detailed understanding of the murine system. In addition, retroviral gene transfer into cultured mouse bone marrow cells has been made possible. While it has been possible to transfer retroviral genes into cultured mouse bone marrow cells, the efficiency of gene transfer into human bone marrow cells has been disappointing to date, which may reflect the fact that human long-term bone marrow cultures have been limited both in their longevity and more importantly in their ability to maintain hematopoietic progenitor cell survival and pluripotentiality over time.
Human bone marrow cultures initially were found to have a limited hematopoietic potential, producing decreasing numbers of hematopoietic progenitor and mature blood cells, with cell production ceasing by six to eight weeks. Subsequent modifications of the original system resulted only in minor improvements. This has been largely attributed to the dependence of the hematopoietic progenitor cells upon environmental influences such essential growth factors (hematopoietic growth factors and cytokines) found in vivo. In addition to these factors, interactions with cell surface molecules and extracellular matrix may be important for hematopoietic progenitor cell survival and proliferation. Previous efforts to advance in vitro proliferation and differentiation of hematopoietic progenitor cells, examined the effects of cytokines in various substrates, including pre-seeded stroma and fibronectin. The addition of exogenous growth factors to the culture environment, particularly IL-3 (Interleukin-3) and GM-CSF (Granulocyte Macrophage-Colony Stimulating Factor), may lead to selective expansion of specific lineages. These findings suggest that addition of exogenous growth factors into hematopoietic progenitor cell cultures may adversely affect the multipotency of primitive hematopoietic progenitor cells by causing them to differentiate and thus depleting the immature hematopoietic progenitor population.
Alternative approaches have used irradiated bone marrow stroma to seed hematopoietic progenitor cells and have been shown to maintain these cells in long-term culture initiating cells (LTCICs) and to increase transduction of hematopoietic progenitor cells and LTCICs by retroviral vectors. However, questions have been raised about the risks of infection and immune reaction to transplantation of non-autologous bone marrow. Fibronectin, a cellular stromal component, reduces this risk of infection and immune mediated response while enhancing retroviral transduction. However, fibronectin alone may not be sufficient to maintain primitive hematopoietic progenitor cells in vitro.
The hypothesis that the three-dimensional micro-environment of the bone marrow plays a role in maintaining hematopoietic stem cell viability and pluripotency has led to investigating structures which mimic this topography. Three-dimensional polymer devices (e.g., nylon mesh) have been shown to support hematopoietic progenitor cell survival, proliferation and multilineage differentiation, but require the presence of growth factors. Such factors can be added exogenously, or supplied via secreting stromal cells which are co-cultured with the progenitor cells, or through the addition of stromal cell conditioned medium.
Hematopoietic progenitor cell expansion for bone marrow transplantation is a potential application of human long-term bone marrow cultures. Human autologous and allogeneic bone marrow transplantation are currently used as therapies for diseases such as leukemia, lymphoma, and other life-threatening diseases. For these procedures, however, a large amount of donor bone marrow must be removed to ensure that there are enough cells for engraftment.
An approach providing hematopoietic progenitor cell expansion would reduce the need for large bone marrow donation and would make possible obtaining a small marrow donation and then expanding the number of progenitor cells in vitro before infusion into the recipient. Also, it is known that a small number of hematopoietic progenitor cells circulate in the blood stream. If these cells could be selected and expanded, then it would be possible to obtain the required number of hematopoietic progenitor cells for transplantation from peripheral blood and eliminate the need for bone marrow donation.
Hematopoietic progenitor cell expansion would also be useful as a supplemental treatment to chemotherapy and is another application for human long-term bone marrow cultures. Most chemotherapy agents act by killing all cells going through cell division. Bone marrow is one of the most prolific tissues in the body and is therefore often the organ that is initially damaged by chemotherapy drugs. The result is that blood cell production is rapidly destroyed during chemotherapy treatment, and chemotherapy must be terminated to allow the hematopoietic system to replenish the blood cell supplies before a patient is re-treated with chemotherapy.
A successful approach providing hematopoietic progenitor cell expansion would greatly facilitate the production of a large number of further differentiated precursor cells of a specific lineage, and in turn provide a larger number of differentiated hematopoietic cells with a wide variety of applications, including blood transfusions.
Gene therapy is a rapidly growing field in medicine with an enormous clinical potential. Traditionally, gene therapy has been defined as a procedure in which an exogenous gene is introduced into the cells of a patient in order to correct an inborn genetic error. Research in gene therapy has been ongoing for several years in several types of cells in vitro and in animal studies, and more recently a number of clinical trials have been initiated.
The human hematopoietic system is an ideal choice for gene therapy in that hematopoietic stem cells are readily accessible for treatment (bone marrow or peripheral blood harvest) and they are believed to possess unlimited self-renewal capabilities (incurring lifetime therapy), and upon reinfusion, can expand and repopulate the marrow. Unfortunately, achieving therapeutic levels of gene transfer into stem cells has yet to be accomplished in humans. The problem which remains to be addressed for successful human gene therapy is the ability to insert the desired therapeutic gene into the chosen cells in a quantity such that it will be beneficial to the patient. To date, methods for the efficient introduction of exogenous genetic material into human hematopoietic stem cells have been limited.
There exists a need to influence favorably hematopoietic progenitor cell viability and pluripotency under long-term culture in vitro.
There exists a need to provide large numbers of differentiated hematopoietic cells.
There also exists the need to improve the efficiency of exogenous genetic material transfer into hematopoietic progenitor cells.
An object of the invention is to provide methods and devices that extend the in vitro viability of hematopoietic stem cells while maintaining the hematopoietic progenitor cell properties of self-renewal and pluripotency.
Another object of the invention is to provide methods and devices for the controlled production in large numbers of specific lineages of progenitor cells and their more differentiated hematopoietic cells.
Yet another object of the invention is to provide improved methods for gene transfer and transduction into cells of hematopoietic origin and hematopoietic progenitor cells in particular. These and other objects of the invention will be described in greater detail below.
The invention, in one important part, involves improved methods for culturing hematopoietic progenitor cells, which methods can, for example, increase the period over which an amount of hematopoietic progenitor cells can be cultured. Thus, one aspect of the invention is improved preservation of a culture of hematopoietic progenitor cells. Another aspect is an improvement in the number of progeny that can be obtained from a sample of hematopoietic progenitor cells. Still another aspect of the invention is an improvement in the number of differentiated progeny blood cells that can be obtained from a sample of hematopoietic progenitor cells.
Surprisingly, according to the invention, it has been discovered that hematopoietic progenitor cells can be cultured without exogenous growth agents for extended periods of time, thereby increasing the supply of hematopoietic progenitor cells and inhibiting the induction of differentiation and/or the loss of progenitor cells during culture. Thus, the present invention permits the culture of hematopoietic progenitor cells in vitro for more than 5 weeks, and even more than 6, 7 or 8 weeks, without adding hematopoietic growth factors, inoculated stromal cells or stromal cell conditioned medium. This is achieved, simply, by culturing the hematopoietic progenitor cells in a porous solid scaffold.
According to one aspect of the invention, a method for in vitro culture of hematopoietic progenitor cells is provided. An amount of hematopoietic progenitor cells is introduced to a porous, solid matrix having interconnected pores of a pore size sufficient to permit the cells to grow throughout the matrix. The cells are cultured upon and within the matrix in an environment that is free of inoculated stromal cells, stromal cell conditioned medium, and exogenously added hematopoietic growth factors that promote hematopoietic cell differentiation, other than serum. The porous matrix can be one that is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. In one embodiment the porous solid matrix has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 xcexcm. Preferably the porous solid matrix is a metal-coated reticulated open cell foam of carbon containing material, the metal coating being selected from the group consisting of tantalum, titanium, platinum (including other metals of the platinum group), niobium, hafnium, tungsten, and combinations thereof. In preferred embodiments, whether the porous solid matrix is metal-coated or not, the matrix is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, functional equivalents of these factors, and combinations thereof. Most preferably the metal coating is tantalum coated with a biological agent. In certain other embodiments the porous solid matrix having seeded hematopoietic progenitor cells and their progeny is impregnated with a gelatinous agent that occupies pores of the matrix.
The preferred embodiments of the invention are solid, unitary macrostructures, i.e. not beads or packed beads. They also involve nonbiodegradable materials.
In other embodiments, before the introducing step, the hematopoietic progenitor cells are obtained from a blood product. Preferably the blood product is unfractionated bone marrow. In still other embodiments, the method further includes the step of harvesting hematopoietic cells. Preferably, there is a first harvesting after a first culturing period and at least one additional harvesting after at least one additional culturing period. The harvested cells then are cultured in at least one of an exogenously added agent selected from the group consisting of a hematopoietic growth factor that promotes hematopoietic cell maintenance, expansion and/or differentiation, inoculated stromal cells, and stromal cell conditioned medium.
According to any of the foregoing embodiments, the method of the invention can include, in said first culturing step, culturing the cells in an environment that is free of hematopoietic progenitor cell survival and proliferation factors such as interleukins 3, 6 and 11, stem cell ligand and FLT-3 ligand. As mentioned above, the inventors have discovered, surprisingly, that hematopoietic progenitor cells can be grown for extended periods of time without the addition of any of these agents which typically are added in the prior art in order to prevent the hematopoietic progenitor cells from dying within several weeks. Still another embodiment of the invention is performing the first culturing step in an environment that is free altogether of any exogenously added hematopoietic progenitor cell growth factors, other than serum.
As will be understood, according to the invention, it is possible now to culture hematopoietic progenitor cells for 6, 7 or 8 weeks, and to harvest hematopoietic progenitor cells during this time interval for subsequent exposure to culture conditions containing hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation. Culturing and harvesting over this time period is an independent aspect of the invention.
According to another aspect of the invention, a method is provided for in vitro culture of hematopoietic progenitor cells to produce differentiated cells of hematopoietic origin. In a first culturing step, a first amount of hematopoietic progenitor cells is cultured in an environment that is free of inoculated stromal cells, stromal cell condition medium and exogenously added hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation, other than serum, under conditions and for a period of time to increase the number of cultured hematopoietic progenitor cells relative to said first amount or to increase the functionality of the hematopoietic progenitor cells, thereby producing a second amount of hematopoietic progenitor cells. Then, in a second culturing step, at least a portion of the second amount of cultured hematopoietic progenitor cells is cultured in an environment that includes at least one of an agent selected from the group consisting of a hematopoietic growth factor that promotes hematopoietic cell maintenance, expansion and/or differentiation, inoculated stromal cells and stromal cell conditioned medium, to produce differentiated cells of hematopoietic origin. In one embodiment, the environment is free of hematopoietic growth factors that promote survival and proliferation of hematopoietic progenitor cells such as interleukins 3, 6 and 11, stem cell ligand and FLT-3 ligand. In another embodiment, the environment of the first culturing step is free of any hematopoietic growth factors other than those present as a result of the addition of serum to the nutritive medium. In this aspect of the invention, the method further can comprise a second culturing step which is a plurality of second culturing steps, each comprising culturing only a portion of the second amount of hematopoietic progenitor cells. The method also can involve a harvesting step between the first and second culturing steps, wherein the harvesting step comprises harvesting the at least a portion of the second amount prior to culturing the at least a portion of the second amount in the second culturing step. The harvesting step also can be a plurality of harvesting steps spaced apart in time and, in this instance, the second culturing step can be a plurality of second culturing steps, one for each of the harvesting steps. The preferred source of the hematopoietic progenitor cells and the preferred configuration of the porous solid matrix is as described above.
According to another aspect of the invention, a method is provided for in vitro culture of hematopoietic progenitor cells to produce differentiated cells of hematopoietic origin. In a first culturing step, hematopoietic progenitor cells are cultured in an environment that is free of inoculated stromal cells, stromal cell condition medium and exogenously added hematopoietic growth factors that promote differentiation, other than serum, to generate cultured hematopoietic progenitor cells. A portion of the cultured hematopoietic progenitor cells are harvested intermittently to generate a plurality of intermittently harvested portions of cultured hematopoietic cells. Then, in a plurality of second culturing steps, the plurality of intermittently cultured harvested portions are cultured in an environment that includes at least one agent selected from the group consisting of a hematopoietic growth factor that promotes differentiation, inoculated stromal cells and stromal cell conditioned medium, to produce differentiated cells of hematopoietic origin. In one embodiment, the environment of the first culturing step is free of hematopoietic growth factors that promote survival and proliferation of hematopoietic progenitor cells, such as interleukins 3, 6 and 11, stem cell ligand and FLT-3 ligand. In another embodiment, the environment of the first culturing step is free of any hematopoietic growth factors, other than those present as a result of the addition of serum to the nutritive medium. In this aspect of the invention, the preferred source of hematopoietic progenitor cells and the preferred porous solid matrix are as described above.
According to another aspect of the invention, a method is provided for transducing exogenous genetic material into cells of hematopoietic origin. Hematopoietic cells are cultured in a porous solid matrix having interconnected pores of a pore size sufficient to permit the cells to grow throughout the matrix. The cells are transduced with the exogenous genetic material in situ on and within the matrix. It has been found, surprisingly, that the efficiency of transfer of genetic material when carried out with the cells cultured upon the matrix is unexpectedly increased. The characteristics of various embodiments of the preferred porous solid matrices are as described above. Also, in this embodiment, the hematopoietic cells can be hematopoietic progenitor cells and the cells, whether progenitor or not, can be cultured in environments free of factors that promote differentiation, factors that promote survival and proliferation, any hematopoietic growth factors whatsoever, inoculated stromal cells or stromal cell conditioned media.
According to still another aspect of the invention, an apparatus for culturing cells is provided. The apparatus includes a first cell culture chamber containing a porous solid matrix having interconnected pores of a pore size sufficient to permit cells to grow throughout the matrix. The apparatus also includes a second cell culture chamber. A conduit provides fluid communication between the first and second cell culture chambers. A collection chamber is located between the first and second cell culture chambers, the collection chamber interrupting fluid communication between the first and second cell culture chambers via the conduit. A first inlet valve on one side of the collection chamber is for providing fluid to be received from the first cultured chamber into the collection chamber. An outlet valve on the other side of the collection chamber provides fluid to be received into the second cultured chamber from the collection chamber. Finally, there is a second inlet valve for the collection chamber for introducing a desired fluid into the collection chamber, other than fluid from the first cell culture chamber, whereby fluid may be intermittently removed from the first cell culture chamber and provided to the second cell culture chamber without contamination of the first culture chamber by fluid from the second culture chamber.
According to yet another aspect of the invention, another apparatus for culturing cells is provided. This apparatus includes a first cell culture chamber containing a porous solid matrix having interconnected pores of a pore size sufficient to permit cells to grow throughout the matrix. An inlet valve on the first cell culture chamber is provided for introducing culture medium into the first cell culture chamber. A second cell culture chamber also is provided, the first and second cell culture chambers being in fluid communication with one another via a conduit. A valve on the conduit is provided for controlling fluid flow between the first and second cell culture chambers.
In either of the foregoing apparatus, the second cell culture chamber can be provided with a porous solid matrix having interconnected pores of a pore size sufficient to permit cells to grow throughout the matrix. Various embodiments are provided, wherein the porous solid matrix has one or more of the preferred characteristics as described above. In addition, the various cell culture chambers can have ports and conduits for sampling material within the cell culture chamber, for augmentation by delivery of various agents to one or the other of the cell culture chambers and for controlling and permitting the continuous flow of medium through either or both of the cell culture chambers.
In yet another aspect of the invention, a solid porous matrix is provided wherein hematopoietic progenitor cells, with or without their progeny, are attached to the solid porous matrix. In some embodiments, stromal cells may also be attached to the matrix. The porous matrix can be one that is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. In one embodiment the porous solid matrix has pores defined by interconnecting ligaments having a diameter at midpoint, on average, of less than 150 xcexcm. Preferably the porous solid matrix is a metal-coated reticulated open cell foam of carbon containing material, the metal coating being selected from the group consisting of tantalum, titanium, platinum (including other metals of the platinum group), niobium, hafnium, tungsten, and combinations thereof. In preferred embodiments, whether the porous solid matrix is metal-coated or not, the matrix is coated with a biological agent selected from the group consisting of collagens, fibronectins, laminins, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogen, antibodies and fragments thereof, functional equivalents of these factors, and combinations thereof. Most preferably the metal coating is tantalum coated with a biological agent. In certain other embodiments the porous solid matrix having seeded hematopoietic progenitor cells and their progeny is impregnated with a gelatinous agent that occupies pores of the matrix.
According to another aspect of the invention, a method for in vivo maintenance, expansion and/or differentiation of hematopoietic progenitor cells is provided. The method involves implanting into a subject a porous, solid matrix having pre-seeded hematopoietic progenitor cells and hematopoietic progenitor cell progeny. The porous matrix has interconnected pores of a pore size sufficient to permit the cells to grow throughout the matrix and is an open cell porous matrix having a percent open space of at least 50%, and preferably at least 75%. Various embodiments are provided, wherein the porous solid matrix has one or more of the preferred characteristics as described above. In certain other embodiments, the porous solid matrix further comprises hematopoietic progenitor cells and their progeny which are attached to the matrix by introducing in vitro an amount of hematopoietic progenitor cells into the porous solid matrix, and culturing the hematopoietic progenitor cells in an environment that is free of inoculated stromal cells, stromal cell conditioned medium, and exogenously added hematopoietic growth factors that promote hematopoietic cell maintenance, expansion and/or differentiation, other than serum. In yet other embodiments the porous solid matrix having seeded hematopoietic progenitor cells and their progeny is impregnated with a gelatinous agent that occupies pores of the matrix.
In any of the foregoing embodiments involving hematopoietic cell maintenance, expansion and/or differentiation using a hematopoietic growth factor, the hematopoietic growth factor used is selected from the group consisting of interleukin 3, interleukin 6, interleukin 7, interleukin 11, interleukin 12 stem cell factor, FLK-2 ligand, FLT-2 ligand, Epo, Tpo, GMCSF, GCSF, Oncostatin M, and MCSF.
These and other aspects of the invention are described in greater detail below.